IMMUNOLOCALIZATION OF INHIBIN/ACTIVIN SUBUNITS AND STEROIDOGENIC ENZYMES IN THE TESTES OF AN ADULT AFRICAN ELEPHANT (LOXODONTA AFRICANA).

In this case report, the authors investigated immunolocalization of inhibin α and inhibin/activin ßA and ßB subunits, as well as steroidogenic enzymes, in the testes of an African elephant. Testes were collected from a reproductively active male African elephant (24 yr old) at autopsy. Histologically, all types of spermatogenic cells including mature-phase spermatozoa were found in the seminiferous tubules. Positive immunostaining for inhibin α and inhibin/activin ßA and ßB subunits was observed in Sertoli and Leydig cells. In addition, P450scc, 3ßHSD, P450c17, and P450arom were also detected in the cytoplasm of Leydig cells. These results suggested that Leydig cells of adult African elephant testes have the ability to synthesize progestin, androgen, and estrogen, whereas both Sertoli and Leydig cells appear as a major source of inhibin secretion in the male African elephant.

Secretory pattern of inhibin during estrous cycle and pregnancy in African (Loxodonta africana) and Asian (Elephas maximus) elephants.

The ovary of female elephants has multiple corpora lutea (CL) during the estrous cycle and gestation. The previous reports clearly demonstrated that inhibin was secreted from lutein cells as well as granulosa cells of antral follicles in cyclic Asian elephants. The aim of this study is to investigate the inhibin secretion during the pregnancy in African and Asian elephants. Two African elephants and two Asian elephants were subjected to this study. Circulating levels of immunoreactive (ir-) inhibin and progesterone were measured by radioimmunoassay. Four pregnant periods of an African elephant and three pregnant periods of an Asian elephant were analyzed in this study. Circulating levels of ir-inhibin started to increase at 1 or 2 week before the ovulation and reached the peak level 3 or 4 weeks earlier than progesterone during the estrous cycle in both African and Asian elephants. After last luteal phase, the serum levels of ir-inhibin remained low throughout pregnancy in both an African and an Asian elephant. The mean levels of ir-inhibin during the pregnancy were lower than the luteal phase in the estrous cycle despite high progesterone levels were maintained throughout the pregnancy. These results strongly suggest that CL secrete a large amount of progesterone but not inhibin during the pregnancy in elephants.

The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants.

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.

Role of the double luteinizing hormone peak, luteinizing follicles, and the secretion of inhibin for dominant follicle selection in Asian elephants (Elephas maximus).

Elephants express two luteinizing hormone (LH) peaks timed 3 wk apart during the follicular phase. This is in marked contrast with the classic mammalian estrous cycle model with its single, ovulation-inducing LH peak. It is not clear why ovulation and a rise in progesterone only occur after the second LH peak in elephants. However, by combining ovarian ultrasound and hormone measurements in five Asian elephants (Elephas maximus), we have found a novel strategy for dominant follicle selection and luteal tissue accumulation. Two distinct waves of follicles develop during the follicular phase, each of which is terminated by an LH peak. At the first (anovulatory) LH surge, the largest follicles measure between 10 and 19.0 mm. At 7 ± 2.4 days before the second (ovulatory) LH surge, luteinization of these large follicles occurs. Simultaneously with luteinized follicle (LUF) formation, immunoreactive (ir) inhibin concentrations rise and stay elevated for 41.8 ± 5.8 days after ovulation and the subsequent rise in progesterone. We have found a significant relationship between LUF diameter and serum ir-inhibin level (r(2) = 0.82, P < 0.001). The results indicate that circulating ir-inhibin concentrations are derived from the luteinized granulosa cells of LUFs. Therefore, it appears that the development of LUFs is a precondition for inhibin secretion, which in turn impacts the selection of the ovulatory follicle. Only now, a single dominant follicle may deviate from the second follicular wave and ovulate after the second LH peak. Thus, elephants have evolved a different strategy for corpus luteum formation and selection of the ovulatory follicle as compared with other mammals.

Secretion of inhibin during the estrous cycle in the female Asian elephant (Elephas maximus).

To define the source of circulating inhibin in female Asian elephants, the immunolocalizations of the inhibin α, ß(A) and ß(B) subunits, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), aromatase cytochrome P450 (P450arom) and cytochrome 17α-hydroxylase P450 (P450 c17) were investigated. Concentrations of immunoreactive (ir-) inhibin, progesterone and follicle-stimulating hormone (FSH) during the estrous cycle were measured by radioimmunoassay. Inhibin immunoreactivity in follicular fluid and homogenate of corpora lutea was also measured. Immunolocalizations of inhibin subunits, 3ß-HSD, P450arom and P450c17 were detected in the granulosa cells of antral follicles and luteal cells. The follicular fluid contained high levels of ir-inhibin and bioactive inhibin. The homogenate of corpora lutea also contained ir-inhibin. Serum ir-inhibin remained at low levels during the early non-luteal phase, began to increase from the late non-luteal phase and continued to increase during the early luteal phase. Serum ir-inhibin showed maximal levels in the middle of the luteal phase and gradually decreased to baseline three weeks prior to progesterone decline. The serum ir-inhibin levels were positively correlated with progesterone throughout the estrous cycle. On the other hand, ir-inhibin was negatively correlated with FSH during the late non-luteal and early luteal phases. These findings strongly suggest that the corpus luteum is one of the sources of inhibin as well as granulosa cells in the Asian elephant.

A surge-like increase in luteinizing hormone preceding musth in a captive bull African elephant (Loxodonta africana).

This study was conducted to determine the correlation between reproductive hormones and musth in a male African elephant. Changes in circulating luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone and immunoreactive (ir-) inhibin and the degree of musth were evaluated for 4 years. LH increased 4 weeks before musth began. The highest concentrations of testosterone and ir-inhibin were observed from April to October. There were positive correlations among testosterone, ir-inhibin and musth behavior. These findings suggested that the surge-like LH in the pre-musth period might stimulate secretion of testosterone and ir-inhibin and thus initiate the musth behavior. This study also suggested that the high LH level before musth might be a useful biomarker for the beginning of the musth season.

Prolactin secretion and ovarian function in cycling and non-cycling African female elephants (Loxodonta africana).

Reproduction of captive elephants in zoos has shown a low fecundity and requires improvement. One of the reasons for low fecundity is ovarian dysfunction in many female elephants. To investigate whether prolactin has a correlation with ovarian function in female elephants, the serum concentrations of prolactin, progesterone and estradiol-17beta in four African female elephants (one cycling female and three non-cycling female elephants) were measured. Cyclic patterns of prolactin and estradiol-17beta were observed in the cycling female elephant, which tended to be high during the follicular phase and low during the luteal phase. On the other hand, a cyclic pattern of prolactin was not observed in the non-cycling female elephants. One of the three non-cycling females (Mako) had developed breasts and showed significantly higher average levels of prolactin than the other female elephants. These results suggested that high concentrations of circulating estradiol-17beta during the follicular phase stimulated prolactin secretion. They also suggested that hyperprolactinemia in Mako was one of the causes of the developed mammary glands and ovarian dysfunction.